Invasive fungal infections are an important cause of morbidity and mortality for people with compromised immunity. Unfortunately, the mortality associated with invasive fungal infections remains unacceptably high. One of the contributing factors to this poor outcome is the fact that there are relatively few therapeutic options for the treatment of invasive fungal infections, particularly when compared to the number of antibiotics available for the treatment of bacterial infections. To identify new antifungal drug candidates, we have initiated a high throughput screening and chemical biology-based project to identify molecules that interfere with fungal cell wall biosynthesis. Application of this strategy has rapidly led to the identification of human phosphoinositide dependent kinase-1 (PDK1) inhibitors as highly active antifungal molecules in vitro. PDK1 inhibitors have been extensively developed as targeted anticancer molecules because of their low toxicity toward normal cells. Encouragingly, two of the PDK1 inhibitors identified in our preliminary work (UCN-01 and OSU-03012) have been, or are being, evaluated in human clinical trials. In order to develop the promising potential of PDK1/Pkh inhibitors as antifungal drugs, we will: characterize the molecular basis for the activity of mammalian PDK1 inhibitors toward fungal PDK1 orthologs (Aim 1); characterize the function of PDK1 orthologs in the human fungal pathogens Candida albicans and Cryptococcus neoformans (Aim 2); determine in vivo efficacy of PDK1 inhibitors in animal models (Aim 3); and optimize antifungal properties of PDK1 inhibitors and identify antifungal PDK1 inhibitors with novel chemical scaffolds (Aim 4). Successful execution of these aims is expected to not only provide a fundamental chemical and biological foundation for the development of antifungal PDK1 molecules but could also lead to the rapid translation of PDK1 inhibitors to clinical use since two of our lead compounds have already been evaluated in human clinical trials.